The migration of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of many vascular diseases. We have previously shown that in vitro VSMC migration in response to PDGF is suppressed in differentiated VSMCs and seek to identify differences in intracellular signalling between differentiated and proliferating VSMCs that may account for this suppression. Differentiated VSMCs retain their ability to respond to PDGF and upregulate expression of the immediate early response genes, c-fos and MCP-1 (JE) when stimulated by PDGF. Unlike proliferating cells, however, PDGF-stimulated differentiated VSMCs fail to activate calcium/calmodulin-dependent protein kinase (CamKinase) II activity. Blocking CamKinase II activation blocked the migration of proliferating VSMCs by more than 90%. In contrast, inhibitors of protein kinase C have no significant effect on migration. Pretreatment of differentiated cells with ionomycin (1 uM) or endothelin (10-100 nM) (both of which are expected to increase intracellular calcium) resulted in an 84 + 6% return to the migration rate of proliferating VSMCs. This return was also blocked by CamKinase inhibitors and was unaffected by inhibitors of PKC. These results suggest that activation of CamKinase plays an important role in VSMC migration and the failure to activate it in differentiated VSMCs may be responsible for the suppression of migration. The most direct intracellular pathway by which PDGF could activate CamKinase II is through the activation of phospholipase Cg (via tyrosine phosphorylation) following its association with the activated PDGF receptor. Immunoprecipitation of PLCg followed by blotting of the protein with an antibody to phosphotyrosine residues indicates that, in contrast to proliferating VSMCs, differentiated VSCMs do, in fact, fail to activate PLCg. Recent experiments have identified a role for basicFGF in the migration of VSMCs and suggest that PLCg activated by PDGF may be indirect and mediated through the FGF receptor. Our results focussing on PDGF intracellular signalling have identified at least one critical difference in the way in which proliferating and differentiated VSMCs respond to PDGF and have demonstrated that to activate differentiated VSMCs requires concurrent action by at least two growth factors/cytokines. This requirement may limit the response of VSMCs to injury to selected group capable of responding to agents.